This invention concerns with a method and a dry analytical element for the determination of bicarbonate ion existing in liquid sample. In particular, the method and dry analytical element of this invention are useful in the determination of the bicarbonate ion in the liquid sample, such as blood or urine, in the clinical test where quick and accurate measurement is required.
A conventional method for the determination of bicarbonate ion existing in liquid sample is of measuring the partial pressure of carbonic acid in liquid and hydrogen ion concentration (pH) by using electrodes. The concentration of bicarbonate ion can be determined by calculating from the above values. However, this method is disadvantageous in the necessity of simultaneous measurement of both the partial pressure of carboxylic acid and the pH of the liquid.
Another conventional method is of utilizing the conversion of bicarbonate ion into carbon dioxide in acidic conditions, and measuring the volume of evolved carbon dioxide. In general, large-scale equipment is necessary for measuring the volume of gas precisely, and accordingly, this method is disadvantageous to the measurement of large number of samples.
In order to improve these disadvantages, some enzyme methods were developed. The enzyme method disclosed in Japanese Patent KOKAI 4-210599 utilizes the following reactions: 
and bicarbonate ion is determined by measuring the decrease of absorption at 340 nm of NAD(P).
In the above described equation, PEPC stands for phosphenolpyruvate carboxylase, PEP stands for phosphenolpyruvic acid, MDH stands for malate dehydrogenase, NADH stands for the reduced form of nicotinamide adenine dinucleotide, NAD(P)H stands for the reduced form of nicotinamide adenine dinucleotide phosphate, and both AND and NADP stands for the oxidized form of nicotinamide adenine dinucleotide and the oxidized form of nicotinamide adenine dinucleotide phosphate respectively.
The method disclosed in Japanese Patent KOKAI 4-248997 uses phosphenolpyruvate carboxykinase instead of PEPC.
However, the above-described methods have following disadvantages:
{circle around (1)} Since in order to measure the absorbance at 340 nm, the analyzer must be equipped with ultra-violet light source and detecting system for the ultra-violet ray. Therefore, the analyzer becomes large-scale, and expensive.
{circle around (2)} Considering reaction rate, since the absorbance of NAD(P)H at 340 nm is of large amount, it is difficult to incorporate a necessary amount of NAD(P)H for converting the whole oxaloacetic acid produced in the above described reaction into malic acid from start. As a result, the determination range becomes narrow.
{circle around (3)} Although the above mentioned large absorbance can be avoided by the change of the measuring wave length to a lower absorbance range, such as 380 nm, and incorporating a sufficient amount of NAD(P)H, the measurement becomes unstable because the spectrum is not flat but oblique.
An object of the invention is to improve the above defects in the conventional determination methods of bicarbonate ion existing in liquid sample. More specifically, it is an object of this invention to provide a reaction system that enables to measure quickly, simply, and stably the bicarbonate ion in liquid using compact instrument with visual light source and sufficient substrate density. Another important object of the present invention is to provide dry analytical element to determine the bicarbonate ion in liquid.
This invention has been made in order to solve the above problems, and the object has been achieved by a method and a dry analytical element for the determination of bicarbonate ion in liquid using phosphenolpyruvate carboxylase and malate dehydrogenase as conjugate enzyme wherein thioNAD(P)H and NAD(P)H are used as substrate of malate dehydrogenase.
In the above described reaction system, reaction proceeds as follows: 
Since thioNAD(P)H has an absorption peak at 400 nm, a visible light source and detection system can be applied.
In the case where only the thioNAD(P)H is used as the substrate, the determination range becomes narrow because the absorbance of the thioNAD(P)H is as high as NAD(P)H. However, this phenomenon can be avoided maintaining the use of the visible light source and spreading the determination range by reducing the quantitative combining ratio of the bicarbonate ion to the amount of the NAD(P)H.
It is not clear what sort of competing reaction occur when bicarbonate ion contacts with thioNAD(P)H and NAD(P)H, but surprisingly the result of measurement about many specimen containing bicarbonate ion of various kinds of concentration showed reproducibility in wide determination range. Therefore, the second disadvantage described above has been solved.